吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (4): 883-891.doi: 10.13481/j.1671-587X.20220406

• 基础研究 • 上一篇    下一篇

兔脱细胞软骨基质颗粒复合SD大鼠脂肪干细胞对软骨内成骨的促进作用

边东潇1,2,包幸福1,2,胡敏1()   

  1. 1.吉林大学口腔医院正畸科,吉林 长春 130021
    2.吉林省牙发育及颌骨重塑与再生重点实验室,吉林 长春 130021
  • 收稿日期:2022-01-16 出版日期:2022-07-28 发布日期:2022-07-26
  • 通讯作者: 胡敏 E-mail:humin@jlu.edu.cn
  • 作者简介:边东潇(1994-),女,吉林省长春市人,在读硕士研究生,主要从事干细胞和骨组织再生方面的研究。
  • 基金资助:
    国家自然科学基金项目(81870795);吉林省科技厅自然科学基金项目(20200201307JC)

Promotion effect of rabbit acellular cartilage matrix particles combined with SD rat adipose tissue-derived stem cells on endochondral osteogenesis

Dongxiao BIAN1,2,Xingfu BAO1,2,Min HU1()   

  1. 1.Department of Orthodontics, Stomatology Hospital, Jilin University, Changchun 130021, China
    2.Jilin Provincial Key Laboratory of Tooth Development and Jaw Bone Remodeling and Regeneration, Changchun 130021, China
  • Received:2022-01-16 Online:2022-07-28 Published:2022-07-26
  • Contact: Min HU E-mail:humin@jlu.edu.cn

摘要: 目的

探讨兔脱细胞软骨基质颗粒(ACM)的生物相容性,阐明其对SD大鼠脂肪干细胞(ADSCs)软骨内成骨的促进作用。

方法

提取原代SD大鼠ADSCs进行三向分化实验,流式细胞术检测细胞干性。采集3月龄新西兰白兔耳软骨,采用研磨浸泡法制备ACM备用。实验分为ACM组和ADSCs+ACM共培养组,在扫描电镜下观察ACM和ACM上ADSCs的超微形态表现,采用钙黄绿素/碘化丙啶 (Calcein-AM/PI)荧光染色法观察ADSCs+ACM共培养组中ADSCs的生长情况。将ADSCs分为对照组和实验组(ADSCs与ACM共培养),采用CCK-8法检测2组细胞增殖活性,实时荧光定量PCR (RT-qPCR) 法检测成软骨诱导7和14 d时2组细胞中Ⅱ型胶原(COL-Ⅱ)、Ⅹ型胶原(COL-Ⅹ)、SOX转录因子9(SOX-9)、血管内皮生长因子(VEGF)、碱性磷酸酶(ALP)和Runt相关转录因子2(RUNX-2)mRNA表达水平。

结果

成功制备兔ACM,提取的SD大鼠ADSCs可以进行三向分化且表达干细胞特异性表面标志物。ADSCs可以在ACM上黏附生长。与对照组比较,实验组ADSCs增殖活性明显升高(P<0.05)。软骨诱导7 d时,与对照组比较,实验组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显升高(P<0.05),COL-Ⅱ、SOX-9和COL-Ⅹ mRNA表达水平差异无统计学意义(P>0.05);软骨诱导14 d时,与对照组比较,实验组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显升高(P<0.05),COL-Ⅹ mRNA表达水平明显降低(P<0.05),COL-Ⅱ和SOX-9 mRNA表达水平差异无统计学意义(P>0.05);与软骨诱导7 d时比较,软骨诱导14 d时对照组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显降低(P<0.05),COL-Ⅱ和COL-Ⅹ mRNA表达水平明显升高(P<0.05);与软骨诱导7 d时比较,软骨诱导14 d时实验组ADSCs中COL-Ⅱ和COL-Ⅹ mRNA表达水平明显升高(P<0.05),VEGF、ALP和RUNX-2 mRNA表达水平明显降低(P<0.05)。

结论

兔ACM缺乏免疫原性,具有良好的生物相容性和成软骨诱导能力,有利于软骨内成骨过程的发生。

关键词: 脂肪干细胞, 脱细胞软骨基质颗粒, 骨再生, 生物材料

Abstract: Objective

To investigate the biocompatibility of rabbit acellular cartilage matrix particles (ACM), and to clarify its promotion effect on the endochondral osteogenesis of adipose tissue-derived stem cells (ADSCs) in the SD rats.

Methods

The primary ADSCs of SD rabbits were extracted for three-dimensional differentiation and flow cytometry was used to detect the cell dryness.The ear cartilage of 3-month-old New Zealand white rabbits was collected and treated with grinding and soaking method to prepare ACM. The experiment was divided into ACM and ACM+ADSCs co-culture group.The ultrastructures of ACM and the ADSCs in ACM were observed under scanning electron microscope,and the growth status of ADSCs in ADSCs+ACM co-culture group was by Calcein-AM/PI fluorescence staining method. The ADSCs were divided into control group and experimental group(ADSCs and ACM co-culture).The CCK-8 method was used to detect the proliferation activities of cells in two groups. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect expression levels of type Ⅱ collagen (COL-Ⅱ),type Ⅹ collagen (COL-Ⅹ), SRY-related high mobility group-box 9 (SOX-9),vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX-2) mRNA in the ADSCs in two groups at 7 and 14 d of chondrogenic induction.

Results

The rabbit ACM was successfully prepared. The extracted ADSCs could differentiate in three ways and the specific surface markers of stem cells were expressed in the ADSCs. The ADSCs could adhere to ACM. Compared with control group, the proliferation activity of the cells in experimental group was increased (P<0.05).The RT-qPCR results showed that compared with control group, the expression levels of VEGF,ALP and RUNX-2 mRNA in the ADSCs in experimental group were increased at 7 d of chondrogentic induction (P<0.05), and the expression levels of COL-2, SOX-9, and COL-Ⅱ mRNA had no significant differences(P>0.05). At 14 d of chondrogentic induction, compared with control group, the expression levels of VEGF, ALP, and RUNX-2 mRNA in the ADSCs in experimental group were increased (P<0.05),and the expression level of COL-Ⅹ mRNA was decreased (P<0.05). Compared with 7 d of chodrogenic induction, the expression levels of VEGF, ALP, and RUNX-2 mRNA in the ADSCs in control group at 14 d of chodrogenic induction were decreased (P<0.05), and the expression levels of COL-Ⅱ and COL-Ⅹ mRNA were increased (P<0.05). Compared with 7 d of chondrogenic induction, the expression levels of COL-Ⅱ and COL-Ⅹ mRNA in the ADSCs in experimental group were increased at 14 d of chondrogenic induction (P<0.05), while the expression levels of VEGF, ALP, and RUNX-2 mRNA were decreased(P<0.05).

Conclusion

The rabbit ACM lack immunogenicity and have good biocompatibility and chondrogenic induction ability, which is beneficial to the occurrence of endochondral osteogenesis.

Key words: Adipose tissue-derived stem cells, Acellular cartilage matrix particles, Bone regeneration, Biomaterial

中图分类号: 

  • Q813.1